Fenretinide (4-HPR) is a synthetic retinoid that is cytotoxic for tumor cells in a p53-independent manner and neuroblastoma cell lines resistant to 13-cis-retinoic acid (13-cis-RA) are highly sensitive to 4-HPR, suggesting 4-HPR may have clinical activity against tumor cells escaping standard 13-cis-RA therapy. We have reported that 4-HPR increased ceramides (via stimulation of de novo synthesis) and that cytoxicity was synergistically enhanced by the ceramide modulators, safingol (L-threo-sphinganine), and PPMP (an inhibitor of glucosyl- and acylceramide synthases). A better understanding of the mechanisms of action of these novel drug combinations, and assessing activity and pharmacokinetics in animal models, will facilitate their clinical development. We hypothesize that a major portion of 4- HPR and 4-HPR + safingol or PPMP is dependent on the ability of 4-HPR to stimulate cfe novo ceramide synthesis. We will use anti-sense and/or siRNA to serine palmitoyl transferase (SPT), the rate-limiting step in ceramide synthesis, to define the role of ceramides in cytotoxicity of these agents (Specific Aim 1) and the molecular ordering of events leading to cell death (Specific Aim 2). Because myc overexpression (MCYN or c-myc) appears to increase cytotoxicity of 4-HPR and retinoic acid, which can decrease MYCN expression, antagonizes 4-HPR in some neuroblastoma cell lines, we hypothesize that high myc levels may facilitate 4-HPR cytotoxicity. In Specific Aim 3 we will use forced overepxression of inducible MYCN or c-myc to determine the influence of myc expression on the cytotoxic response to 4-HPR, safingol, and PPMP. We will determine if antagonism of 4-HPR by retinoic acid is mediated via retinoid receptors and/or associated with decreased MYCN expression. Finally, we hypothesize that with appropriate formulations, 4-HPR + safingol and/or PPMP will deliver tumor-effective drug levels to tissues and will have anti-neuroblastoma activity in vivo with tolerable systemic toxicity. We will test this hypothesis in animal models and clinical trials in Specific Aim 4. We have developed (via RAID and FLAIR grant support) new parenteral formulations suitable for intravenous infusion in humans and for treating mouse xenografts (by i.p. injection) with 4-HPR + safingol and/or PPMP. We have also developed a pediatric oral 4-HPR formulation. We will study these drugs in various combinations and dose schedules for activity against established tumors and minimal residual disease of human neuroblastomas xenografted in immunocompromised mice. Pharmacology (plasma and intracellular drug levels) will be carried out for both the preclinical studies and for our planned phase I clinical trials to enable us to design preclinical models that better reflect the clinical situation. In summary, the studies proposed here will further our understanding of the mechanism of action and activity in vivo of 4-HPR + ceramide modulators in neuroblastoma and will facilitate the design of future phase I, II, and III clinical trials of these druas for neuroblastoma and likelv for other childhood and adult cancers.